Protein folding, the final step in the cellular pathway that translates the information stored in a DNA sequence into a heteropolymeric molecule with a structure-defined function, is of such paramount importance for cell survival that quality-control mechanisms have evolved to ensure native structures form and that misfolded proteins are repaired or targeted for disposal. As might be expected for such a vital process, incorrect and inefficient protein folding are associated with a large number of diseases. Novel drugs for protein conformational diseases could be directed at repairing or rescuing misfolded proteins. Rational design of these compounds depends on fundamental understanding of folding and misfolding mechanisms. The objective of this research program is the elucidation of the conformational changes that transform disordered polypeptides into properly folded functional proteins. Four specific aims involve experimental studies intended first to identify, and then to modulate, the folding mechanisms in a set of helical-bundle proteins. Fluorescence energy transfer methods will be used in Aim 1 to define distributions of distances between pairs of residues in helical-bundle-heme proteins as they fold. These distributions will provide low-resolution structural maps of helical-bundle cytochromes as they form native structures. Aim 2 involves joint experimental/computational studies of the folding mechanism of acyl coenzyme A binding protein. Distance distributions for multiple donor-acceptor pairs placed throughout the protein will be compared directly to computational results. Data from experiments will refine computational models, and output from simulations will aid experimental designs by directing probe placement. The incorporation of fluorinated nonpolar amino acids into proteins has the potential to dramatically alter refolding pathways. Work on Aim 3 involves placement of fluorinated leucine, isoleucine, and valine residues at selected locations in four-helix-bundle proteins with the aim of optimizing and redirecting native folding pathways. Combined energy- and electron-transfer methods will be used in Aim 4 to evaluate the diffusive dynamics in nonnative polypeptide structures. The objective is to determine whether differences in folding rates of topologically similar proteins arise from differences in diffusion dynamics. Designing new therapeutic agents for protein conformational diseases requires knowledge of how proteins fold and misfold. A clear understanding of the dynamics of polypeptide conformational motions is the first step on the pathway to new molecules that can help proteins avoid or escape from the incorrect structures that lead to disease.